La(3+) and Gd(3+) induce shape change of giant unilamellar vesicles of phosphatidylcholine

Lanthanides such as La(3+) and Gd(3+) are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La(3+) and Gd(3+) on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 microM La(3+) (or Gd(3+)) through a 10-microm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 microM) of La(3+) (or Gd(3+)) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 microM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L(beta') to P(beta') phase transition temperature of DPPC-MLV increased with an increase in La(3+) concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La(3+) concentration. Thereby, the interaction of La(3+) (or Gd(3+)) on the external monolayer membrane of the GUV induces a decrease in its area (A(ex)), whereas the area of the internal monolayer membrane (A(in)) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (DeltaA=A(ex)-A(in)).     

Multiple forms of α2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): striking sequence diversity in functional sites

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.     

Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth.

BACKGROUND: Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses. MATERIALS AND METHODS: We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique. RESULTS: We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation. CONCLUSION: Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.     

The Time Course of Dynamic Computed Tomographic Appearance of Radiation Injury to the Cirrhotic Liver Following Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma

This study aimed to evaluate the dynamic computed tomographic (CT) appearance of focal radiation injury to cirrhotic liver tissue around the tumor following stereotactic body radiation therapy (SBRT) for hepatocellular carcinoma (HCC). Seventy-seven patients with 92 HCCs were observed for >6 months. Sixty-four and 13 patients belonged to Child–Pugh class A and B, respectively. The median SBRT dose was 48 Gy/4fr. Dynamic CT scans were performed in non–enhanced, arterial, portal, and venous phases. The median follow-up period was 18 months. Dynamic CT appearances were classified into 3 types: type 1, hyperdensity in all enhanced phases; type 2, hypodensity in arterial and portal phases; type 3, isodensity in all enhanced phases. Half of the type 2 or 3 appearances significantly changed to type 1, particularly in patients belonging to Child–Pugh class A. After 3–6 months, Child–Pugh class B was a significant factor in type 3 patients. Thus, dynamic CT appearances were classified into 3 patterns and significantly changed over time into the enhancement group (type 1) in most patients belonging to Child–Pugh class A. Child–Pugh class B was a significant factor in the non–enhancement group (type 3).     

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.     

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulates synaptic vesicle release

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.     

Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes

Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Recent studies place altered mitochondrial oxidative phosphorylation (OxPhos) as an underlying genetic element of insulin resistance. However, the causative or compensatory nature of these OxPhos changes has yet to be proven. Here, we show that muscle- and liver-specific AIF ablation in mice initiates a pattern of OxPhos deficiency closely mimicking that of human insulin resistance, and contrary to current expectations, results in increased glucose tolerance, reduced fat mass, and increased insulin sensitivity. These results are maintained upon high-fat feeding and in both genetic mosaic and ubiquitous OxPhos-deficient mutants. Importantly, the effects of AIF on glucose metabolism are acutely inducible and reversible. These findings establish that tissue-specific as well as global OxPhos defects in mice can counteract the development of insulin resistance, diabetes, and obesity.     

La autoantigen translocates to cytoplasm after cleavage during granzyme B-mediated cytotoxicity

Our recent report demonstrated that apoptosis-specific autoantibodies against granzyme B-induced cleavage fragments of SS-B (La) were found in the sera from patients with primary Sj?gren's syndrome. The objective of this study was identified by the intracellular redistribution of La autoantigen during granzyme B-induced apoptosis. We developed green fluorescence protein (GFP)-La and GFP-LaDelta220 (generation of granzyme B-specific cleavage of La protein) fusion proteins. GFP-La protein was localized in the nucleus, whereas the GFP-LaDelta220 protein predominantly existed in the cytoplasm in transformed A293T cells. Nuclear GFP-La protein was translocated to cytoplasm after granzyme B enriched YT cells incubation. La protein in human salivary grand HSG cells is cleaved and translocated from the nucleus to the cytoplasm after YT cell co-cultivation. These results suggest that La protein is cleaved by granzyme B and N-terminal La fragment (27 kD) translocated to the cytoplasm, thus leading to a novel autoantibody production during granzyme B-mediated cytotoxicity.